High concentration antibody-containing liquid formulation

ABSTRACT

The problem to be solved is to provide a humanized anti-IL-6 receptor antibody MRA-containing formulation which is suitable for subcutaneous administration, wherein dimerization or deamidation is prevented during long-term storage. The present application is directed to a stable antibody-containing liquid formulation characterized by containing arginine and histidine buffer. A method of inhibiting deamidation or dimerization of such an antibody in a concentrated liquid formulation includes histidine buffer in the liquid formulation.

TECHNICAL FIELD

The present invention relates to an antibody-containing formulation, andparticularly, to a stable liquid formulation containing a highconcentration of an antibody.

BACKGROUND ART

In recent years, various antibody formulations have been developed andused in practice. Many such antibody formulations are used inintravenous injection. However, due to needs of a clinical site, thereis an increasing demand for development of an antibody-containingformulation that can be administered as a self-injectable subcutaneousinjection.

In designing an antibody-containing formulation for subcutaneousinjection, since a dose of an antibody per administration is large(about 100 mg to 200 mg) and an amount of an injection solution isgenerally limited in subcutaneous injection, it is necessary to increasea concentration of an antibody in a liquid to be administered. In viewof this, in many cases, high concentration formulations are used, whichare prepared by the lyophilization-concentration technique, in which alyophilized formulation is reconstituted in water having a volumesmaller than that before lyophilization. However, a strong demand existsfor a liquid formulation which does not require reconstitution, andwhich is easy to handle. Although an increase in a viscosity of aformulation due to addition of a cryoprotective agent such as a sugar inthe production process of the lyophilized formulation is not preferredfor formulations for subcutaneous injection, it is surmised that thisproblem could be avoided if the formulation were a liquid formulation.

Solutions containing a high concentration of an antibody tend to formsolutions having a high viscosity due to macromolecular properties ofproteins, and due to the intermolecular interactions of proteins.Further, in cases where a protein is stored in a form of a solutionhaving a high concentration, problematic degradation occurs, whichincludes a generation of insoluble and/or soluble aggregates; and it isnecessary to prevent such degradation. Especially, in antibodyformulations, associations are likely to be formed and insolubleaggregates are likely to be generated in a liquid state. In cases wherea liquid formulation is stored for a long time, a problem exists in thata bioactivity of antibody molecules is lost due to deamidation of aminoacid residues such as asparagine residues.

There have been proposed various ideas for providing a stabilizedformulation, in which loss of an active component is small even afterthe formulation is stored for a long period of time. Such formulationsare produced by dissolving an active component and various additives ina buffer solution. However, for liquid formulations containing a highconcentration of an antibody, there does not yet exist a technology thatis sufficient to prevent dimerization and deamidation.

A need to provide a high concentration antibody-containing formulationexists, in which dimerization and deamidation during long-term storageare inhibited, and which is both stable and suitable for use insubcutaneous administration.

DISCLOSURE OF THE INVENTION Problem to Be Solved by the Invention

An object of the present invention is to provide a high concentrationantibody-containing liquid formulation, in which dimerization anddeamidation during long-term storage are inhibited, and which is stableand suitable for use in subcutaneous administration.

Means for Solving the Problem

The present inventors conducted intensive study with a view to attainingthe above object, and as a result, discovered that a stable highconcentration antibody-containing liquid formulation can be provided byadding an amino acid, arginine or a salt thereof, as a stabilizer, tothereby complete the present invention.

That is, the present invention provides the following:

-   (1) A stable antibody-containing liquid formulation, characterized    by comprising arginine and methionine.-   (2) The formulation of (1) further comprising a histidine buffering    agent.-   (3) The formulation of (1) or (2) further comprising a surfactant.-   (4) The formulation according to (1) to (3) containing the antibody    in an amount of at least 50 mg/ml.-   (5) The formulation according to (1) to (3) containing the antibody    in an amount of at least 100 mg/ml.-   (6) The formulation according to (1) to (3) containing the antibody    in an amount of at least 120 mg/ml.-   (7) The formulation according to (1) to (6) wherein the antibody is    an anti-IL-6 receptor antibody.-   (8) A stable liquid formulation containing an anti-IL-6 receptor    antibody, characterized by comprising either arginine or methionine.-   (9) The formulation according to (1) to (8) wherein the antibody is    a humanized antibody or human antibody.-   (10) The formulation according to (1) to (9) further comprising    tryptophane.-   (11) The formulation according to (1) to (10) having the pH in the    range from 4 to 8.-   (12) The formulation according to (1) to (11) wherein the arginine    is present in an amount of from 50 to 1500 mM.-   (13) The formulation according to (1) to (12) having a viscosity of    from 2 to 15 mPa·s.-   (14) The formulation according to (1) to (13), which is stable at    22-28° C. for at least 6 months.-   (15) The formulation according to (1) to (13), characterized in that    dimerization of antibody molecules is inhibited.-   (16) The formulation according to (1) to (13), characterized in that    deamidation of antibody molecules is inhibited.-   (17) The formulation according to (1) to (13), which is for    subcutaneous administration.-   (18) The formulation according to (1) to (13) which has not been    subjected to lyophillization during preparation of the formulation.-   (19) A method for inhibiting deamidation of molecules of an antibody    in a liquid formulation containing the antibody, comprising adding    arginine to the liquid formulation.-   (20) A method for inhibiting dimerization of molecules of an    antibody in a liquid formulation containing the antibody, comprising    adding arginine and methionine to the liquid formulation.

Advantages of the Invention

By the present invention, a liquid formulation containing a highconcentration of an antibody is provided, with which reformulation byconcentration by lyophilization is not necessary, and hence does notrequire reconstitution. The antibody-containing liquid formulationaccording to the present invention can be stored in a liquid state for along time. Since the antibody-containing liquid formulation according tothe present invention can be produced by a process not including alyophilization step, addition of a sugar or the like as a cryoprotectiveagent is not necessary.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a typical chromatogram of Example 1.

FIG. 2 shows evaluation results of the gel permeation chromatography(SEC) in Example 1.

FIG. 3 shows evaluation results of the gel permeation chromatography(SEC) in Example 1.

FIG. 4 shows a typical chromatogram of Example 2.

FIG. 5 shows evaluation results of the ion exchange chromatography (lEC)in Example 2.

FIG. 6 shows evaluation results of the ion exchange chromatography (IEC)in Example 2.

FIG. 7 shows evaluation results of the gel permeation chromatography(SEC) in Example 3.

FIG. 8 shows evaluation results of the ion exchange chromatography (IEC)in Example 3.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be described in detail.

In the present invention, “antibody-containing liquid formulation” meansa liquid formulation containing an antibody as an active component,which is prepared such that it can be administered to an animal such ashuman, and which is preferably produced by a process not including alyophilization step.

The antibody-containing liquid formulation according to the presentinvention is a liquid pharmaceutical formulation containing an antibodyat a high concentration, which preferably has an antibody concentrationof not less than 50 mg/mL, more preferably not less than 100 mg/mL,still more preferably not less than 120 mg/mL, and yet more preferablynot less than 150 mg/mL. It should be noted that a liquid formulationcontaining antibody at a concentration of 120 mg/mL or higher, orpreferably 150 mg/mL or higher, has not been developed for commercialuse. Namely, the present invention allows for the first time to put touse a liquid formulation containing antibody at this high concentration.

Further, considering the manufacturing process, the highestconcentration of antibody in the liquid formulation according to thepresent invention may be typically 300 mg/mL, preferably 250 mg/mL andmore preferably 200 mg/mL. Therefore, the antibody-containing liquidformulation according to the present invention preferably has anantibody concentration of from 50 to 300 mg/mL, more preferably from 100to 300 mg/mL, still more preferably from 120 to 250 mg/mL, and yet morepreferably from 150 to 200 mg/mL.

The antibody to be used in the present invention is not restricted aslong as it binds to a desired antigen. The antibody can be either apolyclonal antibody or a monoclonal antibody, although a monoclonalantibody is preferred because an antibody having uniform properties canbe produced stably.

A monoclonal antibody which can be used in the present inventionincludes not only monoclonal antibodies originated from an animal suchas human, mouse, rat, hamster, rabbit, sheep, camel or monkey, but alsoincludes artificially modified recombinant antibodies such as chimericantibody, humanized antibody and bispecific antibody. The immunoglobulinclass of the antibody is not restricted, and can be any of the classesincluding IgGs such as IgGl, IgG2, IgG3 and IgG4, IgA, IgD, IgE and IgM.Among these classes, IgG and IgM are preferred.

The antibody which can be used in the present invention includes notonly whole antibodies, but also antibody fragments such as Fv, Fab andF(ab)₂; and low molecular weight antibodies such as single chain Fv(scFv, sc(Fv)₂, diabodies such as scFv dimer) having one or morespecificities, prepared by binding the variable regions of an antibodythrough a linker such as a peptide linker.

The above-described antibodies which can be used in the presentinvention can be prepared by methods well known to those skilled in theart.

A hybridoma producing a monoclonal antibody can be prepared as followsbasically utilizing a known technique. That is, the hybridoma can beprepared by immunizing an animal with a desired antigen or cellsexpressing the desired antigen as a sensitizing antigen by a standardmethod; fusing the obtained immunocytes with known parent cells by astandard cell-fusion method; and screening a monoclonalantibody-producing cell (hybridoma) by a standard screening method.Preparation of a hybridoma can be carried out by, for example, themethod according to the method by Milstein et al (Kohler. G. andMilstein, C., Methods Enzymol. (1981) 73: 3-46). In cases where theimmunogenicity of the antigen is low, the antigen can be bound to anantigenic macromolecule such as albumin, and the resulting conjugate canbe used as an immunogen.

Recombinant antibodies can be employed, which are prepared by thegenetic recombination technique in which an antigen gene is cloned froma hybridoma, incorporating the gene into an appropriate vector,introducing the vector into a host, and making the host produce theantibody (see, for example, Carl, A. K. Borrebaeck, James, W. Larrick,THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom byMACMILLAN PUBLISHERS LTD, 1990). More specifically, a cDNA encoding thevariable region (V region) in the antibody is synthesized from the mRNAof a hybridoma using a reverse transcriptase. If a DNA encoding the Vregion of the desired antibody is obtained, the DNA is then ligated to aDNA encoding the constant region (C region) of a desired antibody, andthe resulting ligated DNA is introduced into an expression vector.Alternatively, a DNA encoding the V region of the antibody can beincorporated into an expression vector containing the DNA encoding the Cregion of the antibody. The DNA is incorporated into the expressionvector such that the DNA is expressed under the control of anexpression-controlling region such as enhancer or promoter. Host cellsare then transformed with the resulting expression vector, and theantibody can be expressed by the host cells.

In the present invention, recombinant antibodies artificially modifiedfor the purpose of reducing the heteroantigenicity to human, such aschimeric antibodies and humanized antibodies can be used. These modifiedantibodies can be produced by known methods. A chimeric antibody is anantibody comprising variable regions in the heavy chain and light chainin an antibody of an animal other than human, such as mouse, andconstant regions in the heavy chain and light chain in an antibody ofhuman, and can be obtained by ligating a DNA encoding the variableregion in the mouse antibody with a DNA encoding the constant region inthe human antibody, incorporating the obtained DNA into an expressionvector, introducing the expression vector into a host, and making thehost produce the antibody.

Humanized antibody is also called reshaped human antibody, and isobtained by transplanting the CDR (complementarity determining region)of, for example, a mouse antibody to the complementarity determiningregion of a human antibody. A standard genetic recombination techniquefor preparing the humanized antibody is also known. Specifically, a DNAdesigned such that the CDR of the mouse antibody and the frameworkregion (FR) of the human antibody are ligated is synthesized by PCRmethod from several oligonucleotides prepared so as to have overlappingregions at their terminals. The obtained DNA is ligated to a DNAencoding the constant region of a human antibody, and the resulting DNAis introduced into an expression vector. The expression vector isintroduced into a host, and the host is made to produce the humanizedantibody (see EP 239400 A and WO 96/02576). As the FR of the humanantibody to be ligated through CDR, one of which complementaritydetermining region forms a good antigen-binding site is selected. Asrequired, an amino acid(s) in the complementarity determining region canbe substituted so that the complementarity determining region of thereshaped human antibody forms an appropriate antigen-binding site (Sato,K. et al., Cancer Res. (1993) 53, 851-856).

Methods for obtaining a human antibody are known in the art. Forexample, a desired human antibody having a binding activity to a desiredantigen can be obtained by sensitizing, in vitro, human lymphocytes withthe desired antigen or with the cells expressing the desired antigen;fusing the sensitized lymphocytes with human myeloma cells, for example,U266 cells; and obtaining the antibody from the cells (see JP 1-59878B). The desired human antibody can also be obtained by immunizing atransgenic animal having all repertories of human antibody genes withthe antigen (see WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO96/34096 and WO 96/33735). Further, a technique by which a humanantibody is obtained by panning using a human antibody library is alsoknown. For example, a variable region of a human body is expressed inthe form of a single chain antibody (scFv) on the surface of a phage byuse of a phage display method, and the phage which binds to the antigencan be selected. By analyzing the gene of the selected phage, the DNAsequence coding for the variable region of the human antibody whichbinds to the antigen can be determined. If the DNA sequence of the scFvwhich binds to the antigen is determined, an appropriate expressionvector containing the sequence is constructed, and the humanizedantibody can be obtained. These methods are well known, and WO 92/01047,WO 92/20791, WO 93/06213, WO93/11236, WO93/19172, WO 95/01438 and WO95/15388 can be referred to.

In cases where an antibody gene is once isolated, and the gene isintroduced into an appropriate host so as to prepare the antibody,appropriate combinations of the host and expression vector can be used.In cases where eukaryotic cells are used as the host, animal cells,plant cells and fungal cells can be used. Known animal cells include (1)mammalian cells, for example, CHO, COS, myeloma, BHK (baby hamsterkidney), Hela and Vero; (2) amphibian cells, for example, Xenopusoocytes and (3) insect cells, for example, sf9, sf21 and Tn5. Knownplant cells include cells originated from plants belonging to the genusNicotiana, for example, Nicotiana tabacum, and the cells can besubjected to callus culture. Known fungal cells include the cellsoriginated from yeasts, for example, those belonging to the genusSaccharomyces such as Saccharomyces cerevisiae; and filamentousbacteria, for example, those belonging to the genus Aspergillus such asAspergillus niger. In cases where prokaryotic cells are used, there areproduction systems using bacterial cells. Known bacterial cells includeE. coli cells and Bacillus subtilis cells. The antibody is obtained byintroducing a desired antibody gene into these cells by transformation,and culturing the transformed cells in vitro.

Antibodies in the form of antibody fragments, low molecular weightantibodies and modified antibodies can also be employed as the antibodyin the present invention. Examples of the antibody fragments and lowmolecular weight antibodies include Fab, F(ab′)₂, Fv, and single chainFv (scFv, sc(Fv)₂ and the like) having one or more specificities,prepared by ligating the Fvs in the H-chain and L-chain through anappropriate linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A.(1988) 85, 5879-5883). Specifically, an antibody is treated with papainor pepsin to generate antibody fragments, or a gene encoding theseantibody fragments is constructed, and the gene is expressed inappropriate host cells after introducing the gene into an expressionvector (see, for example, Co, M. S. et al., T. Immunol.(1994)152,2968-2976 ; Better, M. and Horwitz, A. H., Methods Enzymol.(1989) 178,476-496; Pluckthun, A. and Skerra, A., Methods Enzymol.(1989) 178, 497-515 ; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663 ;Rousseaux, J. et al., Methods Enzymol. (1986)121, 663-669 ; Bird, R. E.and Walker, B. W., Trends Biotechnol. (1991) 9, 132-137.

Antibodies bound to various molecules such as polyethylene glycol (PEG)can also be used as modified antibodies. The term “antibody” used in thepresent invention also includes these modified antibodies. Thesemodified antibodies can be obtained by chemically modifying an obtainedantibody. Methods for carrying out the modifications are established inthe art.

Examples of the antibody contained in the formulation according to thepresent invention include, but not limited to, anti-tissue factorantibodies, anti-IL-6 receptor antibodies, anti-IL-6 antibodies, HM1.24antigen monoclonal antibodies, anti-parathyroid hormone-related peptideantibodies (anti-PTHrP antibodies), anti-glypican-3 antibodies,anti-ganglioside GM3 antibodies, anti-TPO receptor antagonistantibodies, factor VIII-substituting antibodies, anti-CD3 antibodies,anti-CD20 antibodies, anti-GPIIb)/IIIa antibodies, anti-TNF antibodies,anti-CD25 antibodies, anti-EGFR antibodies, anti-Her2/neu antibodies,anti-RSV antibodies, anti-CD33 antibodies, anti-CD52 antibodies,anti-IgE antibodies, anti-CD11a antibodies, anti-VEGF antibodies,anti-VLA4 antibodies, anti-AXL antibodies, and so on.

Preferred examples of the reshaped human antibodies used in the presentinvention include humanized anti-interleukin (IL-6) receptor antibodies(hPM-1 or MRA) (see WO 92-19759), humanized anti-HM1.24 antigenmonoclonal antibodies (see WO 98-14580), humanized anti-parathyroidhormone-related peptide antibodies (anti-PTHrP antibodies) (see WO98-133881), humanized anti-tissue factor antibodies (see WO 99-51743)and anti-glypican-3 humanized IgG1κ antibodies (see PCT/JP05/013103).The humanized antibodies especially preferred in the present inventionare humanized anti-IL-6 receptor antibodies.

As the human IgM antibodies, anti-ganglioside GM3 recombinant human IgMantibodies (see WO 05-05636) and the like are preferred.

As the low molecular weight antibodies, anti-TPO receptor antagonistdiabodies (see WO 02-33072), anti-CD47 agonist diabodies (see WO01-66737) and the like are preferred.

To evaluate the shelf stability of the high concentrationantibody-containing liquid formulation, the present inventors studiedthe effects of various additives by conducting heat acceleration testsand light acceleration tests. As a result, it was found that insolutions in which a high concentration of antibody was dissolved in abuffer solution containing the amino acid arginine, the amount ofgenerated dimer was smaller than that in solutions to which arginine wasnot added. From these results, it was found that arginine is effectiveas a stabilizer for inhibiting dimerization. Further, in solutions inwhich a high concentration of antibody was dissolved in a buffersolution containing arginine and methionine, the inhibitory effectagainst dimerization was observed at a total concentration of arginineand methionine which is lower than the concentration of arginine aloneneeded for attaining the same inhibitory effect. From these results, itwas found that a synergistic effect is obtained by the addition ofarginine and methionine in combination. Further, it was found thatdeamidation of the antibody molecules is inhibited by the addition ofarginine. These results are exemplified as test results obtained for asample containing a humanized anti-IL-6 receptor antibody at aconcentration of 180 mg/ml.

Thus, by adding arginine as a stabilizer, a stable antibody formulationcan be provided, in which dimerization of the antibody is reduced anddeamidation of the antibody is prevented. Therefore, a first aspect ofthe present invention is characterized by adding arginine to a solution,whereby dimerization or deamidation of the antibody molecules isinhibited in the resulting antibody-containing liquid formulation.Accordingly, an embodiment as a stable antibody-containing liquidformulation is characterized in that it contains an antibody andarginine in a buffer solution. Further, as described above, anantibody-containing liquid formulation of the present invention canadditionally contain methionine in the solution, with a synergisticeffect being obtained by use of arginine and methionine in combination.Therefore, a second aspect of the present invention is characterized byadding arginine and methionine to a solution, whereby dimerization, inparticular, of the antibody molecules is inhibited in the resultingantibody-containing liquid formulation. Accordingly, an embodiment as astable antibody-containing liquid formulation is characterized in thatit contains an antibody, arginine and methionine in a buffer solution.

As the arginine used in the present invention, any of the argininecompound per se, derivatives thereof and salts thereof can be used.L-arginine and salts thereof are preferred. As the methionine used inthe present invention, any of the methionine compound per se,derivatives thereof and salts thereof can be used. L-methionine andsalts thereof are preferred.

In cases where the antibody-containing liquid formulation according tothe present invention contains arginine and does not contain methionine,the concentration of arginine is preferably 50 to 1500 mM, morepreferably 100 to 1000 mM, still more preferably 200 to 700 mM. In caseswhere the antibody-containing liquid formulation according to thepresent invention contains arginine and methionine, the totalconcentration of arginine and methionine is preferably 50 to 1200 mM,for example, preferably, the arginine concentration is 40 to 1000 mM andthe methionine concentration is 10 to 200 mM; more preferably, thearginine concentration is 50 to 700 mM and the methionine concentrationis 10 to 100 mM; and still more preferably, the arginine concentrationis 100 to 300 mM, and the methionine concentration is 10 to 50 mM.

The buffer solution is prepared using a buffering agent which is asubstance for maintaining a pH of the solution. In a high concentrationantibody-containing liquid formulation according to the presentinvention, a pH of the formulation is preferably 4 to 8 , morepreferably 5.0 to 7.5, still more preferably 5.5 to 7.2, and still morepreferably 6.0 to 6.5. A buffering agent which can be used in thepresent invention is one which can adjust the pH in this range and whichis pharmaceutically acceptable. Such a buffering agent is known by thoseskilled in the art, and examples thereof include inorganic salts such asphosphoric acid salts (sodium or potassium) and sodium hydrogencarbonate; organic acid salts such as citric acid salts (sodium orpotassium), sodium acetate and sodium succinate; and acids such asphosphoric acid, carbonic acid, citric acid, succinic acid, malic acidand gluconic acid. Further, Tris buffers, Good’s buffers such as MES,MOPS and HEPES, histidine (e.g., histidine hydrochloric acid salt) andglycine can also be used. In the high concentration antibody-containingliquid formulation according to the present invention, the buffer ispreferably a histidine buffer or glycine buffer, and a histidine bufferis especially preferred. The concentration of the buffer solution isgenerally 1 to 500 mM, preferably 5 to 100 mM, still more preferably 10to 20 mM. In cases where a histidine buffer is used, the buffer solutioncontains histidine at a concentration of preferably 5 to 25 mM, morepreferably 10 to 20 mM.

For the “stable” high concentration antibody-containing liquidformulation according to the present invention, significant change isnot observed when it is stored at a refrigeration temperature (2 to 8°C.) for at least 12 months, preferably for 2 years, and more preferablyfor 3 years; or when it is stored at room temperature (22 to 28° C.) forat least 3 months, preferably 6 months, and more preferably 1 year. Forexample, sum amount of dimers and degradation products in theformulation when it is stored at 5° C. for 2 years is 5.0% or lower,preferably 2% or lower, and more preferably 1.5% or lower; or sum amountof dimers and degradation products in the formulation when it is storedat 25° C. for 6 months is 5.0% or lower, preferably 2% or lower, andmore preferably 1.5% or lower.

The formulation according to the present invention can further contain asurfactant.

Typical examples of the surfactant include nonionic surfactants, forexample, sorbitan fatty acid esters such as sorbitan monocaprylate,sorbitan monolaurate and sorbitan monopalmitate; glycerin fatty acidesters such as glycerol monocaprylate, glycerol monomyristate andglycerol monostearate; polyglycerol fatty acid esters such asdecaglyceryl monostearate, decaglyceryl distearate and decaglycerylmonolinoleate; polyoxyethylene sorbitan fatty acid esters such aspolyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitanmonooleate, polyoxyethylenesorbitan monostearate, polyoxyethylenesorbitan monopalmitate, polyoxyethylene sorbitan trioleate andpolyoxyethylene sorbitan tristearate; polyoxyethylene sorbitol fattyacid esters such as polyoxyethylene sorbitol tetrastearate andpolyoxyethylene sorbitol tetra oleate; polyoxyethylene glycerin fattyacid esters such as polyoxyethylene glyceryl monostearate; polyethyleneglycol fatty acid esters such as polyethylene glycol distearate;polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether;polyoxyethylene polyoxypropylene alkyl ethers such as polyoxyethylenepolyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propylether and polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylenealkyl phenyl ethers such as polyoxyethylene nonylphenyl ether;polyoxyethylene hardened castor oils such as polyoxyethylene castor oiland polyoxyethylene hardened castor oil (polyoxyethylene hydrogenatedcastor oil); polyoxyethylene bees wax derivatives such aspolyoxyethylene sorbitol bees wax; polyoxyethylene lanolin derivativessuch as polyoxyethylene lanolin; surfactants having an HLB of 6 to 18such as polyoxyethylene fatty acid amides, for example, polyoxyethyleneoctadecanarrride; anionic surfactants, for example, alkyl sulfate saltshaving a C₁₀-C₁₈ alkyl group, such as sodium cetyl sulfate, sodiumlauryl sulfate and sodium oleyl sulfate; polyoxyethylene alkyl ethersulfate salts in which the average number of moles of the added ethyleneoxide units is 2 to 4 and the number of carbon atoms of the alkyl groupis 10 to 18, such as polyoxyethylene sodium lauryl sulfate; alkylsulfosuccinate salts having a C₈-C₁₈ alkyl group, such as sodium laurylsulfosuccinate; natural surfactants such as lecithin andglycerophospholipids; sphingophospholipids such as sphingomyelin; andsucrose esters of C₁₂-C₁₈ fatty acids. These surfactants can be added tothe formulation of the present invention individually, or two or more ofthese surfactants can be added in combination.

Preferred surfactants are polyoxyethylene sorbitan fatty acid esters andpolyoxyethylene polyoxypropylene alkyl ethers, and especially preferredare polysorbates 20, 21, 40, 60, 65, 80, 81 and 85, and Pluronic ® (highmolecular weight polyoxyalkylene ether) type surfactants, and mostpreferred are polysorbates 20 and 80, and Pluronic ® F-68 (Poloxamer188).

The amount of the surfactant(s) to be added to the antibody formulationaccording to the present invention is generally 0.0001 to 10% (w/v),preferably 0.001 to 5%, more preferably 0.005 to 3%.

In another aspect of the present invention, the formulation according tothe present invention is preferably substantially composed of thefollowing components:

-   A) anti-IL-6 receptor antibody;-   B) arginine and/or methionine, and additional other amino acid(s)    (e.g., tryptophan) as an optional additional component(s);-   C) buffering agent(s); and-   D) surfactant(s).

The term “substantially composed of” herein means that a component otherthan the components usually added to formulations is not contained, thecomponents usually added to formulations being the optional additivecomponents described below, such as suspending agents, solubilizingagents, isotonic agents, preservatives, adsorption inhibitors, diluents,vehicles, pH-adjusters, soothing agents, sulfur-containing reducingagents and antioxidants.

The above-described “B) arginine and/or methionine, and additional otheramino acid(s) (e.g., tryptophan) as an optional additional component(s)”is meant to include the cases where the formulation contains (b-1)arginine; (b-2) arginine and methionine; and (b-3) methionine;respectively, as an amino acid additive(s), and further include thecases where the formulation additionally contains other amino acid(s).Preferred example of the other amino acid(s) is tryptophan. As thetryptophan, any of the tryptophan compound per se, derivatives thereofand salts thereof can be used. L-tryptophan and salts thereof arepreferred.

As required, a suspending agent, solubilizing agent, isotonic agent,preservative, adsorption inhibitor, diluent, vehicle, pH-adjuster,soothing agent, sulfur-containing reducing agent, antioxidant and thelike can be added to the formulation according to the present invention.

Examples of the suspending agent include methyl cellulose, polysorbate80, hydroxyethyl cellulose, gum arabic, powdered tragacanth, sodiumcarboxymethylcellulose and polyoxyethylene sorbitan monolaurate.

Examples of the solubilizing agent include, polyoxyethylene hydrogenatedcastor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitanmonolaurate, macrogol and castor oil fatty acid ethyl ester.

Examples of the isotonic agent include sodium chloride, potassiumchloride and calcium chloride.

Examples of the preservative include methyl p-hydroxybenxoate, ethylp-hydroxybenzoate, sorbic acid, phenol, cresol and chlorocresol.

Examples of the adsorption inhibitor include human serum albumin,lecithin, dextran, ethyleneoxide-propylene oxide copolymer,hydroxypropylcellulose, methyl cellulose, polyoxyethylene hydrogenatedcastor oil and polyethylene glycol.

Examples of the sulfur-containing reducing agent include the compoundshaving a sulfhydryl group(s), such as N-acetylcysteine, N-acetylhomocysteine, thioctic acid, thiodiglycol, thioethanolamine,thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodiumthiosulfate, glutathione and C₁-C₇ thioalkanes.

Examples of the antioxidant include erythorbic acid,dibutylhydroxytoluene, butylated hydroxyanisole, α-tocopherol,tocopherol acetate, L-ascorbic acid and salts thereof, L-ascorbylpalmitate, L-ascorbyl stearate, sodium hydrogen sulfite, sodium sulfite,triamyl gallate, propyl gallate, and chelating agents such as disodiumethylenediaminetetraacetate (EDTA), sodium pyrophosphate and sodiummetaphosphate.

The antibody-containing liquid formulation according to the presentinvention is usually administered through a parenteral route, forexample, by injection (subcutaneous, intravenous, intramuscularinjections or the like), percutaneous, transmucosal, transnasal orpulmonary administration, but it can also be administered orally. Insubcutaneous injection, the dose of antibody per administration is large(about 100 to 200 mg) while the amount of the injection solution islimited, so that the formulation according to the present invention isespecially suited for subcutaneous injection.

The osmotic pressure ratio of the antibody-containing liquid formulationaccording to the present invention is preferably about 0.5 to 4, morepreferably about 0.7 to 2, and still more preferably about 1.

The viscosity of the antibody-containing liquid formulation according tothe present invention is preferably about 2 to 15 mPa·s, more preferablyabout 4 to 10 mPa·s. It should be noted that the viscosity describedherein is measured by a rotation viscometer method using a cone-platetype viscometer, in accordance with 2.53 Viscosity Determination /General Tests, the Japanese Pharmacopoeia, 15th edition.

As can be seen from the results of the examples described below,according to the present invention, a stable liquid formulation can beobtained, in which dimerization and deamidation of the antibody duringlong-term storage are small, by adding to the formulation argininealone, or arginine and methionine, or methionine alone.

As another aspect of the present invention, a method for inhibitingdeamidation in antibody-containing liquid formulations is provided, themethod comprising adding to the formulation arginine or a salt thereof.

As still another aspect of the present invention, a method forinhibiting dimerization of antibody in antibody-containing liquidformulations is provided, the method comprising adding to theformulation arginine and methionine.

In the above-described two methods, the antibody is preferably ananti-IL-6 receptor antibody, which is a humanized antibody or humanantibody.

The present invention will now be described in more detail by way of theexamples given below. However, the scope of the present invention is notrestricted thereto.

EXAMPLES Antibody Sample

The humanized anti-IL-6 receptor antibody was the humanized antibodyprepared in accordance with the method described in Reference Example 2in JP 8-99902 A using the human elongation factor Iα promoter describedin Example 10 in WO 92/19759. This antibody will occasionally bereferred to as “MRA” in the tables in Examples.

Example 1 Stabilizing Effects by Combination of Arginine and Methionine

Liquid formulations containing anti-IL-6 receptor humanized antibodywere evaluated for an influence on stabilization of the formulationsobtained by use of a combination of arginine and methionine.

In this study, to evaluate the effects by the combination of arginineand methionine, evaluation samples numbered A1 to A9 were prepared.Prescriptions for the evaluation samples were as follows:

TABLE 1-1 Prescriptions Sample No. Antibody mg/mL Arg mM Met mMPolysorbate 80 mg/mL Histidine buffer mM pH A1 180 - - 0.5 20 6.0 A2 18050 - 0.5 20 6.0 A3 180 100 - 0.5 20 6.0 A4 180 150 - 0.5 20 6.0 A5 180200 - 0.5 20 6.0 A6 180 300 - 0.5 20 6.0 A7 180 100 10 0.5 20 6.0 A8 180100 30 0.5 20 6.0 A9 180 100 50 0.5 20 6.0

To evaluate stability of the liquid formulations, each sample wassubjected to a heat acceleration test (stored at 40° C. for 3 months andat 25° C. for 6 months, respectively). The purity of the antibody beforeand after the heat acceleration test was evaluated by gel permeationchromatography (SEC). The analytical conditions were as follows:

Gel Permeation Chromatography

The sample was used as the solution to be measured as it was.

One microliter of the solution to be measured was subjected to liquidchromatography, and the peak areas of the peaks of dimer, monomer andlow molecular weight degradation products (LMW) were measured by anautomatic analytical method, and the amounts thereof (%) weredetermined.

TABLE 1-2 Analytical Conditions Column: TSKgel G3000SWx1 7.8 mm I. D. x30 cm (TOSOH) Mobile Phase: phosphate buffer, pH 7.0 (50 mmol/Lphosphate buffer, pH 7.0, containing 300 mmol/L of sodium chloride and0.05% sodium azide) Amount of Injected Sample: about 180 µg in terms ofhumanized anti-IL-6 receptor antibody Flow Rate: 1 mL/min DetectionWavelength: 280 nm

$\begin{array}{l}\text{Calculation Equation} \\\text{Total Area of All Peaks = Peak Area of Monomer + Peak Area} \\\text{of Dimer + Peak Area of Low Molecular Weight Degradation Products} \\\text{(LMW)} \\\text{Amount of Dimer (\%)=(Peak Area of Dimer/Total Area of All} \\{\text{Peaks)} \times \text{100}} \\\text{Amount of Low Molecular Weight Degradation Products (LMW)} \\\text{(\%)=(Peak Area of Low Molecular Weight Degradation Products/} \\{\text{Total Area of All Peaks)} \times \text{100}}\end{array}$

A typical chromatography is shown in FIG. 1 .

The evaluation results obtained by the gel permeation chromatography(SEC) are shown in Table 1 and FIGS. 2 and 3 . As shown, the amount ofdimer in the samples (Sample Nos. A2 to A6) to which arginine was added,after the acceleration at 40° C. for 3 months and at 25° C. for 6months, respectively, was smaller than that in the sample (Sample No.A1) to which arginine was not added; and accordingly, the inhibitoryeffect of arginine against dimerization was confirmed. It was alsoconfirmed that the amount of dimer was reduced proportionally to theamount of the arginine added. On the other hand, the amount of dimer inthe samples (Sample Nos. A7 to A9) to which arginine (100 mM) andmethionine were added, after the acceleration at 40° C. for 3 months andat 25° C. for 6 months, respectively, was smaller than that in thesamples (Sample Nos. A3 and A4) containing 150 mM of arginine, whichconcentration was about the same as the total concentration of thestabilizers; and the amount of dimer was about the same as in the sample(Sample No. A6) having an arginine concentration of 300 mM. Theseresults are considered to indicate that a synergistic effect in theinhibition of dimerization is obtained by combining arginine andmethionine.

Influence of arginine and methionine on the amount of low molecularweight degradation products was not observed.

TABLE 1-3 40° C.-3 months 25° C.-6 months Dimer (%) LMW (%) Dimer (%)LMW (%) A1 2.70 1.25 1.88 0.48 A2 2.19 1.24 1.41 0.47 A3 2.00 1.34 1.330.49 A4 1.85 1.38 1.19 0.49 A5 1.62 1.37 1.09 0.49 A6 1.53 1.46 0.990.50 A7 1.58 1.29 1.11 0.45 A8 1.52 1.21 1.07 0.47 A9 1.48 1.32 1.030.47

Example 2 Inhibitory Effect by Arginine Against Deamidation

Liquid formulations containing and-IL-6 receptor humanized antibody wereevaluated for influence on the deamidation by arginine.

In this study, evaluation samples numbered A10 to A15 and numbered A16to A18, containing different amounts of arginine and methionine,respectively, were prepared. Prescriptions for the evaluation sampleswere as follows:

TABLE 2-1 Prescriptions Sample No. Antibody mg/mL Arg mM Met mMPolysorbate 80 mg/mL Histidine buffer mM pH A10 180 - - 0.5 20 6.0 A11180 50 - 0.5 20 6.0 A12 180 100 - 0.5 20 6.0 A13 180 150 - 0.5 20 6.0A14 180 200 - 0.5 20 6.0 A15 180 300 - 0.5 20 6.0 A16 180 - 10 0.5 206.0 A17 180 - 30 0.5 20 6.0 A18 180 - 50 0.5 20 6.0

To evaluate the stability of the liquid formulations, each sample wassubjected to a heat acceleration test (stored at 40° C. for 3 months andat 25° C. for 6 months, respectively). The purities of the antibodybefore and after the heat acceleration test were evaluated byion-exchange chromatography (TEC). The analytical conditions were asfollows:

Ion-Exchange Chromatography

To each sample, purified water was added to adjust the amount of thehumanized anti-IL-6 receptor antibody to about 1 mg in 1 mL of thesample, and the resulting sample was used as the sample to be measured.

Thirty microliters of the sample solution was subjected to liquidchromatography, and the peak areas of the peaks of MRA Pre, MRA Main,MRA Sub-1,MRA Sub-2, MRA R-1, 1Q(H)-MRA, 2Q(H)-MRA and other relatedsubstances (Others) were measured by an automatic analytical method, andthe amounts thereof (%) were determined by an area percentage method.

MRA Pre indicates the total of the peaks of the substances each elutedafter a retention time shorter than that of the main component, and aplurality of degradation products, mainly deamidation products ofhumanized anti-IL-6 receptor antibody, was included. When the productionamount of this Pre peak was small, inhibition of deamidation of theantibody is indicated.

TABLE 2-2 Analytical Conditions Column: ProPac™ WCX-10 4 x 250 mmchromatography column (DIONEX® separation materials) Mobile Phase:Solution A: 25 mmol/L MES buffer solution, pH 6.1 Mobile Phase: SolutionB: 25 mmol/L MES buffer solution, pH 6.1 (containing 250 mmol/L ofsodium chloride) Amount of Injected Sample: about 30 µg in terms ofhumanized anti-IL-6 receptor anti body Flow Rate: 0.5 mL/min DetectionWavelength: 280 nm

$\begin{array}{l}\text{Calculation Equation} \\\text{Total Area of All Peaks=Grand Total of Total Area of MRA Pre} \\{\text{Peaks + Peak Area of}\text{.MRAMain + Peak Area of MAR Sub-1 + Peak}} \\\text{Area of MAR Sub-2 + Peak Area of MAR Sub-3 + Peak Area of MAR} \\\text{R-1 + Total Area of 1Q(H)-MRA Peaks + Total Area of 2Q(H)-MRA} \\\text{Peaks \_ Peak Area of Others} \\\text{Amount of MRA Pre (\%)=(Total Area of MRA Pre Peaks/Total} \\{\text{Area of All Peaks)} \times \text{100}}\end{array}$

A typical chromatography is shown in FIG. 4 . MRA Pre indicates thetotal of the peaks of the substances appearing earlier than that of themain component.

Evaluation results of the ion-exchange chromatography are shown in Table2 and FIGS. 5 and 6 . As shown, the amount of Pre peaks in the samples(Sample Nos. A11 to A15) to which arginine was added, after theacceleration at 40° C. for 3 months and at 25° C. for 6 months,respectively, was smaller than that in the sample (Sample No. A10) towhich arginine was not added; and accordingly, the inhibitory effect ofarginine against the generation of Pre peaks was confirmed. It was alsoconfirmed that the amount of Pre peaks was reduced proportionally to anamount of arginine added. On the other hand, the amount of Pre peaks inthe samples (Sample Nos. A16 to A18) to which methionine was added,after the acceleration at 40° C. for 3 months and at 25° C. for 6months, respectively, was similar to the sample (Sample No. A10) towhich arginine was not added; and accordingly, influence of the additionof methionine was not observed.

TABLE 2-3 Table 2 Pre peak (%) 40° C.-3 months 25° C.-6 months A10 56.232.3 A11 51.3 30.3 A12 50.7 29.3 A13 49.0 28.7 A14 47.8 28.5 A15 47.027.9 A16 55.7 31.2 A17 55.0 31.2 A18 55.3 31.4

Example 3 Stabilizing Effects by Combination of Arginine and Methionine(2)

As in Example 1, liquid formulations containing anti-IL-6 receptorhumanized antibody were evaluated for influence on stabilization of theformulations obtained by use of a combination of arginine andmethionine.

In this study, to evaluate effects of the combination of arginine andmethionine, evaluation samples numbered A19 to A27 were prepared.Prescriptions for the evaluation samples were as follows:

TABLE 3-1 Prescriptions Sample No. Antibody mg/mL Arg mM Met mMPolysorbate 80 mg/mL Histidine buffer mM pH A19 180 - - 0.5 20 6.0 A20180 50 - 0.5 20 6.0 A21 180 100 - 0.5 20 6.0 A22 180 150 - 0.5 20 6.0A23 180 200 - 0.5 20 6.0 A24 180 300 - 0.5 20 6.0 A25 180 100 10 0.5 206.0 A26 180 100 30 0.5 20 6.0 A27 180 100 50 0.5 20 6.0

To evaluate the stability of the liquid formulations, each sample wassubjected to a light acceleration test (total illuminance 1,200,000 luxand total near-ultraviolet radiation energy: 200 W·h/m²). The puritiesof the antibody before and after the light acceleration test wereevaluated by gel permeation chromatography (SEC) and ion exchangechromatography (IEC) as in Examples 1 and 2.

The evaluation results by the gel permeation chromatography (SEC) areshown in Table 3 and FIG. 7 . As shown, the amount of dimer in thesamples (Sample Nos. A20 to A24) to which arginine was added, after thelight acceleration test was smaller than that in the sample (Sample No.A19) to which arginine was not added; and accordingly, the inhibitoryeffect of arginine against dimerization was confirmed. It was alsoconfirmed that the amount of dimer was reduced proportionally to anamount of arginine added. On the other hand, the amount of dimer in thesamples (Sample Nos. A25 to A27) to which arginine (100 mM) andmethionine were added, after the light acceleration test was smallerthan that in the sample (Sample No. A22) containing 150 mM of arginine,which concentration was about the same as the total concentration of thestabilizers; and the amount of dimer was smaller than in the samples(Sample Nos. A23 and A24) having arginine concentrations of 200 mM and300 mM, respectively. These results are thought to indicate that asynergistic effect in the inhibition of dimerization is obtained bycombining arginine and methionine.

Influence of arginine and methionine on the amount of low molecularweight degradation products was not observed.

TABLE 3-2 Table 3 1,200,000lux + 200 W · h/m²) Dimer (%) LMW (%) A196.95 0.22 A20 6.75 0.24 A21 5.78 0.21 A22 5.08 0.19 A23 4.73 0.18 A244.13 0.18 A25 5.27 0.19 A26 4.05 0.17 A27 3.84 0.16

Next, the evaluation results by the ion exchange chromatography (IEC)are shown in Table 4 and FIG. 8 .

As shown, the amount of Pre peak in the samples (Sample Nos. A20 to A24)to which arginine was added, after the light acceleration test wassmaller than that in the sample (Sample No. A19) to which arginine wasnot added; and accordingly, the inhibitory effect of arginine againstformation of Pre peak was confirmed. Further, it was confirmed that asthe amount of arginine increases, the production amount of Pre peakdecreases proportionately. On the other hand, the amount of dimer afterthe light acceleration test in the samples (Sample Nos. A25 to A27) towhich methionine was further added to arginine (100 mM) was smaller thanthat in the sample (Sample No. A22) containing 150 mM of arginine, whichconcentration was about the same as the total concentration of thestabilizers; and it was smaller than in the samples (Sample Nos. A23 andA24) having arginine concentrations of 200 mM and 300 mM, respectively.These results are thought to indicate that a synergistic effect in theinhibition of formation of Pre peak by the combination of arginine andmethionine.

TABLE 4 Pre peak (%) 1,200,000lux + 200 W. h/m² A19 39.2 A20 38.6 A2136.7 A22 35.7 A23 34.9 A24 34.9 A25 36.8 A26 35.0 A27 33.8

1. A concentrated liquid formulation comprising 180 mg/mL of a humanizedanti-IL-6 receptor antibody MRA, 0.005 to 3% surfactant, and histidinebuffer, pH 6.0, wherein the formulation has no more than 2.70% dimersafter storage at 40° C. for 3 months or no more than 1.88% dimers afterstorage at 25° C. for 6 months.
 2. The formulation of claim 1 furthercomprising arginine.
 3. The formulation of claim 2, wherein the arginineconcentration is 50 to 300 mM.
 4. The formulation of claim 1 furthercomprising methionine.
 5. The formulation of claim 4, wherein themethionine concentration is 10 to 50 mM.
 6. The formulation of claim 1wherein the surfactant is polysorbate 80.